Mutations that restrict replication of dengue virus have been sought for the generation of recombinant live-attenuated dengue virus vaccines. Dengue virus type 4 (DEN4) was previously grown in Vero cells in the presence of 5-fluorouracil, and the characterization of 1,248 mutagenized, Vero cell-passaged clones identified 20 temperature-sensitive (ts) mutant viruses that were attenuated (att) in suckling mouse brain. The present investigation has extended these studies by identifying an additional 22 DEN4 mutant viruses that have a small-plaque size (sp) phenotype in Vero cells and/or the liver cell line, HuH-7. Five mutant viruses have a sp phenotype in both Vero and HuH-7 cells, three of which are also ts. Seventeen mutant viruses have a sp phenotype in only HuH-7 cells, thirteen of which are also ts. Each of the sp viruses was growth restricted in the suckling mouse brain, exhibiting a wide range of reduction in replication (9- to 100,000-fold). Complete nucleotide sequence was determined for the 22 DEN4 sp mutant viruses, and nucleotide substitutions were found in the 3? untranslated region (UTR) as well as in all coding regions except NS4A. Identical mutations have been identified in multiple virus clones suggesting that they may be involved in the adaptation of DEN4 virus to efficient growth in Vero cells. Six of the 22 sp 5-FU mutant viruses lacked coding mutations in the structural genes, and 17 recombinant DEN4 viruses were generated which separately encoded each of the mutations observed in these six sp viruses. Analysis of the recombinant DEN4 viruses defined the genetic basis of the sp, ts and att phenotypes observed in the six sp viruses. Mutations in NS1, NS3, and the 3? UTR were found to confer a greater than 100-fold, 10,000-fold, and 1,000-fold reduction in replication of rDEN4 virus in SCID mice transplanted with HuH-7 cells, respectively, which serves as a novel small animal model for DEN4 infection. Using reverse genetics, it is possible to readily add well-defined attenuating mutations to the genome of wild type or incompletely attenuated dengue (DEN) viruses to generate vaccine candidates that exhibit the desired balance between attenuation and immunogenicity. Here, we describe the identification of eight temperature sensitive missense mutations distributed in four non-structural protein genes that specify a 60- to 10,000-fold range of restricted replication in the suckling mouse brain compared to wild type recombinant DEN4 virus. These eight novel ts att mutations have several attributes that make them attractive for inclusion in a cDNA-based DEN virus vaccine. First, they are distributed throughout the genome in four genes permitting their use in combination with other att mutations in the same gene or in a different gene or UTR. The ability to combine single ts att mutations in multiple genes by reverse genetics has been shown to both increase att phenotypes as well as the genetic stability of virus vaccine strains. The specific combination of the 3575 and 4062 mutations in NS2A was compatible for viability and each independently contributed to the attenuation of the 5FU parent virus, identifying this pair of mutations as a valuable and compatible combination of attenuating mutations. Second, the mutations described here are located outside of the structural gene region of the dengue virus genome and therefore can be inserted into vaccine candidates without modifying the envelope protein, the major protective antigen of the virus that induces neutralizing antibodies. As such, they can be used as attenuating mutations in antigenic chimeric DEN virus vaccines containing a DEN4 genetic background with structural genes from another. Third, a wide range of attenuation in the suckling mouse brain (60-fold to 12,000-fold reduction in replication) was specified by these eight mutations, suggesting that they may be useful in conferring a high level of attenuation on a wild type virus as well as in fine-tuning the level of attenuation of a slightly under-attenuated vaccine candidate, such as 2AD30. Fourth, mutations were identified which selectively confer a ts phenotype in human liver cells (4062 or 5695), and it will be of particular interest to determine whether such mutations might be used to decrease the mild liver toxicity associated with some DEN vaccine strain. Charge-to-alanine mutagenesis of dengue virus type 4 (DEN4) NS5 gene generated a collection of attenuating mutations for potential use in a recombinant live attenuated dengue virus vaccine. Codons for 80 contiguous pairs of charged amino acids in NS5 were individually mutagenized to create uncharged pairs of alanine residues, and 32 recombinant mutant viruses were recovered from the 80 full-length mutant DEN4 cDNA constructs. These mutant viruses were tested for temperature-sensitive (ts) replication in both Vero cells and HuH-7 human hepatoma cells. Thirteen of the 32 mutants were temperature sensitive in both cell lines; 11 were not ts in either cell line; and 8 exhibited a host-range (tshr) phenotype. One tshr mutant was ts only in Vero cells and seven were ts only in HuH-7 cells. Nineteen of the 32 mutants were ten-fold or more restricted in replication in the brain of suckling mice compared to that of wild type DEN4, and three mutants were approximately 10,000-fold restricted in replication. The level of temperature sensitivity of replication in vitro did not correlate with attenuation in vivo. A virus bearing two pairs of charge-to-alanine mutations was constructed and demonstrated increased temperature sensitivity and attenuation relative to either parent virus. This large set of charge-to-alanine mutations specifying a wide range of attenuation for mouse brain should prove useful in fine-tuning recombinant live attenuated dengue virus vaccines. The D30 deletion mutation, originally created in dengue virus type 4 (DEN4) by the removal of nucleotides 172-143 from the 3?-UTR, was introduced into a homologous region of wild type dengue virus type 1 (DEN1). The resulting virus, rDEN1D30, was attenuated in rhesus monkeys to a level similar to that of the rDEN4D30 vaccine candidate. rDEN1D30 was more attenuated in rhesus monkeys than the previously described vaccine candidate, rDEN1mutF, which also contains mutations in the 3?-UTR, and both vaccines were highly protective against challenge with wild type DEN1. Both rDEN1D30 and rDEN1mutF were also attenuated in HuH-7-SCID mice. However, neither rDEN1D30 nor rDEN1mutF showed restricted replication following intrathoracic administration in the mosquito Toxorhynchites splendens. The ability of the D30 mutation to attenuate both DEN1 and DEN4 viruses suggests that a tetravalent DEN vaccine could be generated by introduction of the D30 mutation into wild type DEN viruses belonging to each of the four serotypes.